This project has two major aims: 1) to determine the importance of mitochondrial damage in the cytotoxic action of known anticancer drugs; 2) to exploit the differences between the mitochondria of cancer and normal cells in the development of new anticancer drugs. For these purposes, it was helpful to use Rhodamine-123(Rh-123), a fluorescent dye selectively accumulated in mitochondria. The uptake of Rh-123, a cationic permeant drug, depends upon the mitochondrial membrane potential (inside negative). Since the conditions for staining (lug/ml x 10 mins) are non-toxic, the morphology and membrane potential of mitochondria in living cells can be monitored. We found that many anticancer drugs cause dramatic, albeit reversible, changes in mitochondrial morphology, followed by an irreversible dissipation of the membrane potential which is evident prior to other indications of cell death. The dissipation of the mitochondrial membrane potential correlates with the loss of clonogenic ability. Thus, Rh-123 may provide a rapid alternative to the clonogenic assay as a predictive test for drug sensitivity. I now plan to examine the consequences of the dissipation of the mitochondrial membrane potential and their importance in the cytotoxic action of known anticancer drugs. We also found that cell types differ in their ability to retain Rh-123 in dye-free medium. Most importantly, carcinomas retain Rh-123 longer than normal cells or other tumor types. Perhaps because of this prolonged retention, we found carcinomas to be more sensitive than non-tumorigenic epithelial cells to the toxic effects of Rh-123 (10Hug/ml x 24 hrs.) in-vitro. Furthermore, Rh-123 prolongs survival of mice implanted with carcinomas. Thus, carcinomas exhibit unusual characteristics of Rh-123 retention and sensitivity, which suggest that the mitochondrial membrane potential or protein components of carcinomas are different from those of normal cells. Our future experiments will allow identification of these differences between carcinomas and normal cells and possible exploitation of these differences for cancer chemotherapy.